IAI Accepts, published online ahead of print on 7 July 2014 Infect. Immun. doi:10.1128/IAI.01779-14 Copyright © 2014, American Society for Microbiology. All Rights Reserved.
1
Brucella melitensis invades murine erythrocytes during
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infection
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#
Marie-Alice Vitry, # Delphine Hanot Mambres, # Michaël Deghelt, #Katrin Hack, #Arnaud
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Machelart, @Frédéric Lhomme, £Jean-Marie Vanderwinden, @Marjorie Vermeersch, *Carl De
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Trez, @David Pérez-Morga, #,1 Jean-Jacques Letesson, †,1 Eric Muraille
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#
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Microbiologie, NARILIS, University of Namur (UNamur). Rue de Bruxelles 61, 5000
Unité de Recherche en Biologie des Microorganismes, Laboratoire d’Immunologie et de
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Namur, Belgium.
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@ Center for Microscopy and Molecular Imaging, Université Libre de Bruxelles (ULB),
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Gosselies, Belgium.
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£ Laboratory of Neurophysiology, Université Libre de Bruxelles, Campus Erasme, Bldg. C,
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808 Route de Lennik, 1070 Brussels, Belgium.
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* Department of Molecular and Cellular Interactions, Vlaams Interuniversitair Instituut voor
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Biotechnologie, Vrije Universiteit Brussel, Gebouw E, Verdieping 8, Pleinlaan 2, 1050
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Brussels, Belgium.
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† Laboratoire de Parasitologie. Faculté de Médecine. Route de Lennik 808, 1070 Bruxelles.
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Université Libre de Bruxelles. Belgique.
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Jean-Jacques Letesson and Eric Muraille are joint senior authors on this work.
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Corresponding authors: - Eric Muraille. Mailing address: Laboratoire de Parasitologie. Faculté de médecine. Route de Lennik 808,
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1070 Bruxelles. Université Libre de Bruxelles. Belgium. Fax.32 (0)2 5556128. Email:
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[email protected]
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Key words: Brucella melitensis, erythrocyte, infection
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Running title: Erythrocyte infection by Brucella melitensis
32
1
33 34
ABSTRACT
35 36
Brucella spp are facultative intracellular Gram-negative coccobacilli responsible for
37
brucellosis, a worldwide zoonosis. We observed that Brucella melitensis is able to persist for
38
several weeks in the blood of intraperitoneally infected mice and transferred blood at any time
39
point tested is able to induce infection in naïve recipient mice. Bacterial persistence in the
40
blood is dramatically impaired by specific antibodies induced following Brucella vaccination.
41
In contrast to Bartonella, the Type IV secretion system and flagella expression are not
42
critically required for the persistence of Brucella in blood. ImageStream analysis of blood
43
cells showed that, following a brief extracellular phase, Brucella is mainly associated with the
44
erythrocytes. Examination by confocal microscopy and transmission electron microscopy
45
formally demonstrated that B. melitensis is able to invade erythrocytes in vivo. The bacteria
46
do not seem to multiply in erythrocytes and are found free in the cytoplasm. Our results open
47
up new areas for investigation and should serve in the development of novel strategies for the
48
treatment or prophylaxis of brucellosis. Invasion of erythrocytes could potentially protect the
49
bacterial cells from the host’s immune response, hamper antibiotic treatment and suggests
50
possible Brucella transmission by blood sucking insects in nature.
51 52 53
2
54
INTRODUCTION
55 56
Brucella is a facultative intracellular α-Proteobacteria that causes abortion and
57
infertility in mammals and leads to a debilitating febrile illness in humans. Although it is
58
rarely fatal, human brucellosis can progress into a severe chronic disease if left untreated (1,
59
2). Despite significant progress, the incidence of human brucellosis remains very high in
60
endemic areas (3). Complete eradication of Brucella would be unpractical due to its presence
61
in a large range of wild mammals (4, 5). Moreover, antibiotic treatment is costly and patients
62
frequently suffer from relapse of the bacteria (6, 7). Vaccination actually remains the only
63
rational strategy to confer protection on populations living in endemic countries.
64
Unfortunately, there is currently no available vaccine against human brucellosis as all
65
commercial animal vaccines are based on live attenuated strains of Brucella (8) that are still
66
virulent in humans. Efforts to develop an effective vaccine are impaired by our poor
67
knowledge of the Brucella life cycle in vivo and of the protective immune response induced
68
by infection. In particular, the mechanisms of early Brucella dissemination in vivo remain
69
largely unknown. A prominent characteristic of brucellosis is its long incubation period. The
70
current hypothesis is that this period, corresponding to reduced activation of innate immunity,
71
opens an “immunological window” and gives Brucella the chance to spread throughout the
72
organism to establish a replication niche within phagocytic cells (9).
73
Bacteremia is one major characteristic of human brucellosis (10) and is often
74
associated with an increased risk of relapse (6, 11). Moreover positive blood cultures are
75
synonymous with secondary seeding and development of focal complications of the disease.
76
Bacteremia in mice infected by intraperitoneal injection, the most frequent route of
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experimental infection, has been reported in some studies (12, 13). However, to our
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knowledge, the mechanism of Brucella persistence in the blood has never been investigated.
3
79
In this study, we monitored bacteremia in mice for several weeks after the intraperitoneal
80
inoculation of B. melitensis strain 16M constitutively expressing the mCherry fluorescent
81
tracer. The location of B. melitensis in the blood cells was analyzed by ImageStream analysis
82
and confocal microscopy. We also investigated the impact of type IV secretion system and
83
flagellum deficiencies as well as of specific antibodies on the blood persistence of the
84
bacteria.
85
Our results show that B. melitensis is able to persist for at least two weeks in the blood
86
of infected mice after intraperitoneal inoculation. The bacteria were first located
87
extracellularly but were then mainly found inside erythrocytes. To our knowledge, this
88
particular niche of infection has never been described in brucellosis, although it is relatively
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common in other bacteria of the α-proteobacteria phylum such as Anaplasma (14) and
90
Bartonella (15). Circulating antibodies induced by vaccination drastically reduced blood
91
persistence of the bacteria following challenge.
92
4
MATERIAL AND METHODS
93 94 95
Ethics Statement
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The animal handling and procedures used in this study complied with current
97
European legislation (directive 86/609/EEC) and the corresponding Belgian law “Arrêté royal
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relatif à la protection des animaux d'expérience du 6 avril 2010 publié le 14 mai 2010"
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(Royal Decree on the protection of experimental animals of April 6, 2010, published on May
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14, 2010). The complete protocol was reviewed and approved by the Animal Welfare
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Committee of the University of Namur (UNamur, Belgium) (Permit Number: 05-558).
102 103
Mice and reagents
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MuMT−/− C57BL/6 mice (16) and C57BL/6 mice transgenic for GFP under the control
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of the ubiquitin C promoter (Ubi-GFP) (17) were purchased from The Jackson Laboratory
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(Bar Harbor, ME). Wild type C57BL/6 mice were purchased from Harlan (Bicester, UK) and
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were used as the control. All wild type and deficient mice used in this study were bred in the
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Gosselies animal facility of the Free University of Brussels (ULB, Belgium).
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B. melitensis strain 16M (Biotype1, ATCC 23456) was grown in a biosafety level III
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laboratory facility. Cultures grown overnight under shaking at 37°C in 2YT media (Luria-
111
Bertani broth with double quantity of yeast extract) to stationary phase were washed twice in
112
PBS (3500xg, 10 min.) as described previously (18). When indicated, we used B. melitensis
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strain 16M which stably expresses the mCherry protein (mCherry-Br), a previously described
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rapidly maturing variant of the red fluorescent protein DsRed (19), under the control of a
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strong Brucella spp. promoter named PsojA (20). The construction of the mCherry-Br strain
116
has been described previously in detail (21). We also used the B. melitensis strain 16M
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producing GFP (constructed by transferring the plasmid pBBR1-GFP-SOG (22) by mating),
5
118
the virB mutant B. melitensis strain 16M (deficient for the type IV secretion system, (23)) and
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the fliC mutant B. melitensis strain 16M (deficient for flagellin expression, (24)).
120 121
Mouse infection
122
Mice were injected intraperitoneally (i.p.) with the indicated dose of B. melitensis in
123
500 μl of PBS. Control animals were injected with the same volume of PBS. The infectious
124
doses were validated by plating serial dilutions of inocula. Mice were bled at selected time
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points and sacrificed at the end of the experiment.
126 127
Blood transfer experiment
128
Mice were injected i.p. (500 µl) with fresh blood from infected donor mice. Blood was
129
only diluted 20 fold in PBS before immediate administration. Two weeks later, spleens were
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recovered in PBS/0.1% X-100 triton. Serial dilutions were performed in PBS and plated onto
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2YT medium plates. The CFU counts were recorded after 4 days of culture at 37°C.
132 133
Antibiotic treatment
134
Mice were injected i.p. with 4x104 CFU of B. melitensis or with PBS. After resting for
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3 weeks, antibiotic treatment was administered to both the immunized and control mice for 3
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weeks. The treatment was a combination of rifampicin (12 mg/kg) and streptomycin (450
137
mg/kg) (adapted from (25)) prepared fresh daily and given in the drinking water. An
138
additional treatment was given i.p. and consisted of 5 injections of streptomycin (300 mg/kg)
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throughout the 3 weeks of oral treatment.
140 141
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142
RAW cell infection
143
RAW cells grown in 6-well tissue culture plates were inoculated with mCherry-Br at a
144
multiplicity of infection (MOI) of 300 in 2 ml of cell culture medium (DMEM 10% FBS).
145
Cells were then centrifuged at 1200 g for 10 minutes at 4°C and placed under 5% CO2 at
146
37°C. After 1 h, the cells were washed two times in PBS and incubated further with cell
147
culture medium supplemented with 50 μg/ml gentamicin until the end of the infection time.
148
After the indicated incubation time, the cells were washed three times in PBS, collected and
149
centrifuged at 1200 g for 10 minutes at 4°C. The cells were fixed in 300 μl of PBS/4% PFA
150
for further ImageStream analysis.
151 152
Bacterial count
153
For bacterial counts in the blood, 70 μl of blood were collected from the tail into
154
heparinized capillary tubes at the selected time points and diluted in PBS/0.1% X-100 triton
155
(Sigma). Serial dilutions were performed in PBS and plated onto 2YT media plates. CFUs
156
were counted after 4 days of culture at 37°C.
157
To separate the blood cells from the plasma, 140 μl of blood were collected and
158
diluted 4 times to allow for better separation. Samples were then centrifuged at 250g for 10
159
minutes at room temperature (RT) and each blood fraction was recovered. An additional
160
centrifugation (8000g, 2 minutes, RT) was performed on the supernatant of samples from
161
mice infected with a low dose of bacteria (4x104 CFU). This was necessary to concentrate the
162
bacteria. The supernatant fraction was diluted in PBS when necessary while the pellet fraction
163
was diluted in PBS/0.1% X-100 triton. Each fraction was then plated on 2YT and CFUs were
164
counted after 4 days of culture at 37°C.
165 166
7
Erythrocytes purification
167 168 169
Blood was collected from the tail into heparinized capillary tubes at the selected time
170
points, diluted in PBS and immediately filtered with Plasmodipur Filters (EuroProxima),
171
following the recommendation of the manufacturer. Plasmodipur Filters removes leukocytes
172
from blood (26, 27).
173 174
Cytofluorometric analysis
175
Blood was collected from the tail into heparinized capillary tubes at the selected time
176
points and diluted in PBS. After washing, blood cells were first incubated in saturating doses
177
of purified 2.4G2 (anti-mouse Fc receptor, ATCC) in 200 μl PBS 0.2% BSA 0.02% NaN3
178
(FACS buffer) for 20 minutes on ice to prevent antibody binding to Fc receptor. Blood cells
179
were stained on ice with various fluorescent mAb combinations in FACS buffer and further
180
collected on a FACScalibur cytofluorometer (Becton Dickinson, BD). We purchased the
181
following mAbs from BD Biosciences: Fluorescein (FITC)-coupled 145-2C11 (anti-CD3ε),
182
Phycoerythrin (PE)-coupled 1A8 (anti-Ly-6G) and Alexa647-coupled M5/114.15.2 (anti-
183
MHC-II).
184 185
Immunofluorescence microscopy on fixed bacteria
186
Blood from Ubiquitin-GFP C57BL/6 mice infected with 5x107 CFU of mCherry-Br
187
was diluted 1/1 (without preliminary wash or centrifugation) for 15 min in PBS containing
188
4% paraformaldehyde (pH 7.4) before being smeared on slides. The slides were dried
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overnight at room temperature and then mounted in Fluoro-Gel medium containing DAPI
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nucleic acid stain (Electron Microscopy Sciences, Hatfield, PA). The labeled cells were
191
visualized with an Axiovert M200 inverted microscope (Zeiss, Iena, Germany) equipped with
192
a high-resolution monochrome camera (AxioCam HR, Zeiss). Images (1384x1036 pixels
193
(0.16µm/pixel)) were acquired sequentially for each fluorochrome with an LD-Plan-NeoFluar
8
194
63x/0.75 N.A. dry objective and recorded as eight bit grey level zvi files. Images were
195
exported as TIFF files and figures were performed using Canvas 7 software.
196 197
Immunofluorescence microscopy on live bacteria
198
Wild type C57BL/6 mice were infected with 5x107 CFU of GFP or mCherry
199
expressing Brucella melitensis. Blood samples were harvested at 3h and 24h post infection
200
and immediately diluted 10x in PBS. For the observation, the samples were directly dropped
201
on an agarose pad (solution of 1% agarose in PBS), covered with a coverslip and sealed with
202
VALAP (1/3 Vaseline, 1/3 lanolin and 1/3 paraffin wax). The sealed pad was placed at 4°C
203
for 10 min before imaging with a Nikon 80i (objective Phase contrast 100X, plan Apo)
204
connected to a Hamamatsu ORCA-ER camera in a biosafety level III laboratory facility.
205 206
Multispectral Imaging Flow Cytometry (ImageStream)
207
Events were imaged using multispectral imaging flow cytometry (ImageStream 100,
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Amnis Corp., Wash). At least 60,000 events were collected from each sample (100,000 for
209
blood samples) and the data were analyzed using image analysis software (IDEAS, Amnis
210
Corp., Wash). Bright field images of the cells and bacteria were recorded in channel 2 and
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mCherry bacteria were recorded in channel 4. The acquisition gate included all cells and
212
bacteria and excluded fragments of cells and cell aggregates. Gate R1 was based on the
213
mCherry signal and allowed for the identification of bacteria inside and outside of blood cells.
214
The distinction between overlapping bacteria and extracellular (free or adherent) bacteria was
215
made by a combination of the erosion mask applied on the bright field image and the intensity
216
mask at 50% in the specific mCherry channel and using the intensity feature of IDEAS
217
software. Briefly, a bacterium was tagged as intracellular when its fluorescent image
9
218
overlapped with the bright field image of blood cells. The intracellular or extracellular
219
location of the bacteria was always confirmed by examination of the images.
220 221
Confocal microcopy
222
Confocal analyses were performed with the LSM780 confocal system fitted on an
223
Observer Z1 inverted microscope equipped with an alpha Plan Apochromat 63x/1.46 N.A. oil
224
immersion objective (Zeiss, Iena, Germany). The 488 nm excitation wavelength of the
225
Argon/2 laser, a main dichroic HFT 488 and a band-pass emission filter (BP500-530 nm)
226
were used for selective detection of the eGFP fluorochrome. The 543nm excitation
227
wavelength of the HeNe1 laser, a main dichroic HFT 488/543/633 and a long-pass emission
228
filter (BP580-650 nm) were used for selective detection of the mCherry fluorochrome. Stacks
229
of one micron-thick (i.e. 1 Airy Unit for the red fluorophore) optical sections (volume
230
sampled: 27 x 27 x 5.2 microns, 512 by 512 pixels, scaling: X & Y: 0.053 micron, Z: 0.370
231
micron) were collected sequentially for each fluorochrome throughout the thickness of the
232
object and stored as 16-bits *.lsm files. File Stacks were processed for deconvolution using
233
Huyghens® software (Scientific Volume Imaging BV, Hilversum, The Netherlands) and 3D
234
rendering was performed using Imaris® software (Bitplane AG, Zurich, Switzerland). Images
235
were exported as 8-bit uncompressed *.TIF image files.
236 237
Scanning electron microcopy
238
Samples were fixed overnight at 4°C in 4% PFA, 0.1M cacodylate buffer (pH 7.2),
239
and post-fixed in OsO4 (2%) in the same buffer. After serial dehydration, samples were dried
240
to the critical point and coated with platinum by standard procedures. Observations were
241
made in a Tecnai FEG ESEM QUANTA 200 (FEI) and images were processed by SIS iTEM
242
(Olympus) software.
10
243 244
Transmission electron microcopy
245
Infected red blood cells were fixed overnight in 2.5% glutaraldehyde in cacodylate
246
buffer, postfixed in 2% osmium tetroxide, serially dehydrated in ethanol and embedded in
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epoxy resin (Agar 100 resin, Agar scientific, UK). Ultrathin 70 nm sections were obtained,
248
mounted on copper-formvar-carbon grids (EMS, UK) and stained with uranyl acetate and lead
249
citrate by standard procedures. Observations were made on a Tecnai 10 electron microscope
250
(FEI, Eindhoven) and images were captured with a Veleta CCD camera and processed with
251
AnalySIS and Adobe Photoshop software.
252 253
ELISA
254
Specific murine IgM, IgG1, IgG2a and IgG3 isotypes were determined by enzyme–
255
linked immunosorbent assay (ELISA). Polystyrene plates (Nunc 269620) were coated with
256
heat-killed B. melitensis (107 CFU/ml). After incubation overnight at 4°C, plates were
257
blocked for 2 hours at room temperature (RT) with 200 μl of PBS-3.65% casein. The plates
258
were then incubated for 1 h at RT with 50 μl of serial dilutions of the serum in PBS-3.5%
259
casein. Sera from naive mice were used as the negative control. After 4 washes with PBS,
260
isotype-specific goat anti-mouse horseradish peroxidase conjugates were added (50μl/well) at
261
appropriate dilutions (αIgM from Sigma; LO-MG1-13 HRPO, LO-MG2a-9 HRPO, LO-
262
MG3-13 HRPO from LO-IMEX). After 1 h of incubation at room temperature, the plates
263
were washed 4 times in PBS, and 100 μl/well of substrate solution (BD OptEiA) was added to
264
each well. After 10 minutes of incubation at room temperature in the dark, the enzyme
265
reaction was stopped by adding 25 μl/well of 2N H2SO4, and the absorbance was measured at
266
450 nm.
267
11
268
Statistical analysis
269
We used a (Wilcoxon-) Mann-Whitney test provided by the GraphPad Prism program
270
to statistically analyze our results. Each group of deficient mice was compared to wild type
271
mice. Values of p < 0.05 were considered to represent a significant difference. *, **, ***
272
denote p50% of the bacteria in the “72h blood”
464
group survived to the treatment with 200 µg/ml of gentamicin. In contrast, only around 10%
465
resisted in other groups (data not shown). Gentamicin is generally presented as an antibiotic
466
that only acts on extracellular bacteria but this is an oversimplification. Several studies have
467
demonstrated that gentamicin can kill intracellular bacteria inside macrophages (43). In
468
addition, pharmacokinetic studies have shown that gentamicin slowly penetrates human
469
erythrocytes (44). We suspect that infected erythrocytes may be more fragile and susceptible
470
to lyse during in vitro incubation. This can explain why bacteria, even when located inside
471
erythrocytes, remain partially sensible to gentamicin in our model.
472
In the model of erythrocyte infection by Bartonella spp, blood persistence and
473
erythrocyte invasion have been reported to be drastically reduced in absence of a functional
474
dependent type IV secretion system (29, 30) or flagella (31, 32). In our Brucella model of
475
infection, a bacterial strain deficient for flagella (FliC mutant) was able to persist in the blood
476
at a level that was similar to the wild type strain, thus demonstrating that flagella are not
477
implicated in blood persistence and presumably in erythrocyte invasion. Deficiency for the
478
type IV secretion system (VirB mutant) did not affect early persistence of bacteria in the
479
blood. Only blood CFU levels appeared to be reduced by 1 log at a later time point. As
480
persistence of the VirB mutant in the spleen is known to be reduced at a later time point (data
481
not shown and (23)), we can only conclude that the type IV secretion system is not critically
482
implicated in blood persistence as this strain remains able to persist until 13 days p.i., in
21
483
contrast to the complete inability of Bartonella type IV secretion system mutants to cause
484
bacteremia (29).
485
We also investigated whether the control of B. melitensis persistence in the blood
486
involves circulating antibodies and B cells. Some studies have reported that natural antibodies
487
play an important protective function in the control of early dissemination of viruses
488
(Vesicular Stomatitis Virus, Vaccinia virus, Lymphocytic ChorioMeningitis Virus) and
489
facultative intracellular bacteria (Listeria monocytogenes) (33, 34). Natural antibodies could
490
increase the early trapping of pathogens in secondary lymphoid organs by enhancing
491
phagocytosis. Previously, several groups have analyzed the role of natural antibodies in an
492
experimental model of Brucella, with contradictory results. Natural antibodies have been
493
reported to promote the control of an attenuated Brucella mutant by facilitating its elimination
494
by phagocytosis (45). Many serum transfer experiments have demonstrated that antibodies
495
play a positive role in protecting mice against Brucella infection (46-48). In this study, we
496
were unable to detect Brucella specific antibodies in naïve mice or during the 3 first days p.i.
497
Specific IgM antibodies were first detected at 6 days and IgG antibodies at 28 days, with a
498
predominance of the IgG2a- and IgG3 isotypes. This observation and the fact that the number
499
of bacteria found in the bloodstream during infection appears to be similar in wild type and
500
MuMT-/- mice demonstrated that natural antibodies had no or only a minor impact on
501
Brucella trapping in naïve mice under our experimental conditions. In contrast, we observed
502
that specific circulating antibodies present in vaccinated wild type mice dramatically reduced
503
the bacteria count persisting in the blood after a secondary infection. This suggests that the
504
blockage of Brucella erythrocyte colonization could constitute an important step in the
505
protective immune response against Brucella infection.
506
Although erythrocytes do not appear to constitute a replication niche for Brucella
507
since each infected cell contained only one single bacterium, they could serve as an important
22
508
part of the pathogen reservoir. Given the long lifespan of erythrocytes (49), Brucella could
509
potentially persist hidden in this environment for several months. The undulating fever
510
characteristic of human brucellosis and the high risk of relapse also argue for regularly
511
occurring new bacteremia waves. In this context, the failure to replicate within erythrocytes
512
could be advantageous for Brucella.
513
Of course, our data were obtained in the murine experimental model of infection with
514
Brucella melitensis and their relevance to other Brucella species and in the natural host
515
remains to be demonstrated. However, bacteremia is common in human brucellosis (11) and
516
an in vitro study has reported that Brucella is able to bind to sialic acid residues on human
517
erythrocytes (50), suggesting that erythrocyte invasion could also occur in patients.
518
From an evolutionary point of view, the unexpected ability of Brucella to infect
519
erythrocytes is not so surprising. Indeed, several bacterial pathogens belonging to the α-
520
(Bartonella spp (28), Anaplasma marginale (51)) and γ- (Francisella tularensis (52, 53))
521
proteobacteria phyla persist inside erythrocytes in the bloodstream during infection and are
522
transmitted to the host by ticks or fleas. Numerous old studies (5, 54-57) and a study
523
published in 2013 (58) have documented the presence of Brucella in blood-sucking
524
arthropods (ticks and fleas) under natural conditions. These observations, combined with our
525
demonstration of the ability of B. melitensis to infect erythrocytes in vivo, render plausible the
526
hypothesis that under natural conditions erythrocytes could constitute an alternative reservoir
527
for Brucella and that Brucella could be transmitted to the host by blood-sucking arthropods.
528
We hope that this hypothesis will be investigated in the future.
529
In conclusion, invasion of erythrocytes could constitute a crucial unexpected step that
530
promotes the evasion of host immune defenses. This new trait of Brucella pathogenesis opens
531
up new areas of research. Since erythrocytes are not capable of endocytosis (59), we will now
532
need to define the precise mechanism of Brucella uptake by the cell and identify the virulence
23
533
factors necessary for this process. We have tested several in vitro protocols to infect
534
erythrocytes from naïve mice. Unfortunately, under conditions where RAW cells were fully
535
infected, we never observed erythrocyte infection (data not shown). In addition, it would be
536
interesting to investigate the homing of infected erythrocytes in tissues and whether they
537
trigger a different immune response compared with other infected cells. We are thus left with
538
the exciting challenge of determining the importance of this mechanism to pathogenesis and
539
transmission of the infection.
540
24
541
ACKNOWLEDGEMENTS
542 543
This work was supported by grants from the Fonds National de la Recherche Scientifique
544
(FNRS) (FRSM FNRS convention 3.4.600.06.F, Belgium), from the Communauté Française
545
de Belgique (Action de Recherches Concertées 08/13–015), from the Interuniversity
546
Attraction
547
(http://www.belspo.be/belspo/iap/index_en.stm). E.M. is a Research Associate from the FRS-
548
FNRS (Belgium). M.A.V. and D.H.M hold PhD grants from the FRS-FNRS (Belgium). A.M.
549
and M.D. hold PhD grants from the FRIA (Belgium). The funding agencies played no role in
550
the design of the study, collection and analysis of the data, the decision to publish, or
551
preparation of the manuscript.
Poles
Programme
initiated
by
the
Belgian
Science
Policy
Office
552
25
FIGURE LEGENDS
553 554 555
Figure 1. Persistence of infectious B. melitensis in the blood of mice after i.p. infection.
556
A, B: Wild type C57BL/6 mice were injected i.p. with 4x104, 106 or 5x107 CFU of mCherry-
557
expressing B. melitensis (mCherry-Br) and blood was collected at selected time points. The
558
data represent either the mean +/- SEM of mCherry-Br CFU per ml of blood (A) or the
559
percentage of mice that still had bacteria in the blood (B). The results are representative of
560
two independent experiments. C, Wild type C57BL/6 mice were injected i.p. with 106 CFU of
561
mCherry-Br and blood was collected at 1, 3, 6 and 13 days p.i. Blood was immediately
562
diluted 20x in PBS and transferred by i.p. (500 µl) to recipient wild type C57BL/6 mice. Two
563
weeks after blood transfer, the mice were sacrificed and the spleens were harvested. The data
564
represent the CFU per spleen and are representative of two independent experiments. The
565
grey bars represent the medians.
566 567
Figure 2. Type IV secretion system (VirB) and flagella (FliC) deficient strains of B.
568
melitensis are able to persist in the blood of infected mice until 3 days. Wild type
569
C57BL/6 mice were injected i.p. with 106 CFU of wild type, FliC or VirB strain of B.
570
melitensis and blood was collected at the indicated time points. The data represent the CFU
571
per ml of blood of individual mice and are representative of three independent experiments.
572
The grey bars represent the medians.
573 574
Figure 3. Natural antibodies do not prevent B. melitensis dissemination in the blood
575
whereas vaccine-induced specific antibodies reduce the bacteremia following a
576
secondary infection. A, Wild type and MuMT-/- C57BL/6 mice were immunized i.p. with
577
4x104 CFU of B. melitensis (vaccinated group) or injected i.p. with PBS (naïve group) and
26
578
then treated with antibiotics for three weeks, as described in the Material and Methods
579
section. 60 days after immunization, all mice were injected i.p. with 5x107 CFU of mCherry-
580
Br and blood was collected at the indicated time points. The data represent the CFU per ml of
581
blood and are representative of three independent experiments. The grey bars represent the
582
medians. B, Wild-type C57BL/6 mice were infected with 4x104 CFU of B. melitensis. Serum
583
was collected at the indicated time points and ELISA was performed to determine the isotype
584
distribution of the Brucella-specific antibodies. The data represent the mean +/- SEM of 6
585
mice.
586 587
Figure 4. Blood location of B. melitensis after i.p. infection. A. Wild type C57BL/6 mice
588
were injected i.p. with 4x104 or 106 CFU of mCherry-Br and blood was collected at the
589
indicated time points. Samples were diluted and centrifuged to separate blood cells from
590
plasma. The data represent the mean +/- SD of the percentage of CFU in each blood fraction
591
and are representative of two independent experiments. B,C. Wild type C57BL/6 mice were
592
injected i.p. with 5x107 CFU of mCherry-Br and blood was collected at the indicated time
593
points. Samples were diluted and immediately filtered with plasmodipur filters. B. Total
594
blood cells and purified erythrocytes are analyzed by flow cytometry using anti-CD3ε, anti-
595
Ly-6G and anti-MHC-II antibodies in order to confirm the elimination of all leukocytes. C.
596
The data represent the CFU per ml of total blood or purified erythrocytes and are
597
representative of two independent experiments with 3 mice per group.
598 599
Figure 5. Immunofluorescence microscopy of fixed or live bacteria. A: Ubiquitin-GFP
600
C57BL/6 mice were infected i.p. with 5x107 CFU of mCherry-Br and bled at 3h and 24h p.i.
601
The blood was fixed and then smeared on slides. The slides were dried overnight and then
602
mounted in Fluoro-Gel medium containing DAPI nucleic acid stain (Electron Microscopy
27
603
Sciences, Hatfield, PA) and examined under a fluorescent microscope. Brucella was found
604
associated with erythrocytes mainly at 24h, and thus we present only images starting at 24h
605
p.i. The figure shows examples of mCherry bacteria associated with DAPI- erythrocytes. The
606
panels are color-coded according to the antigen examined. The scale bar = 20 or 10 µm, as
607
indicated. The data are representative of at least 3 independent experiments with a total of
608
more 200 bacteria observed. B, C: Wild type C57BL/6 mice were infected with 5x107 CFU of
609
mCherry-Br (B) or GFP-Br (C). Blood samples were harvested at 3h and 24h p.i. and
610
immediately diluted 10x in PBS. Freshly harvested blood samples were directly dropped on
611
an agarose pad and sealed with VALAP before observation under a fluorescent microscope in
612
a biosafety level III laboratory facility. The data are representative of at least 3 independent
613
experiments with a total of more than 30 bacteria observed under each condition.
614 615
Figure 6. ImageStream analysis of blood from infected mice
616
Wild type C57BL/6 Mice were injected i.p. with 5x107 CFU of mCherry-Br and blood was
617
collected at 3h, 24h and 72h p.i. The blood samples were immediately fixed by 3-fold dilution
618
in 4% paraformaldehyde and then analyzed by multispectral imaging flow cytometry
619
(ImageStream 100). As controls, we used a fresh culture of wild type Brucella, mCherry-Br
620
and a macrophage cell line (RAW cells) at 3h post in vitro infection with mCherry-Br. A.
621
Comparison of the percentage of events displaying a positive mCherry (channel 4) fluorescent
622
signal (gate R1) in blood from control and infected mice. B. Representative image of free
623
bacteria, uninfected and infected cells in channels 2 and 4. C, D. Kinetic analysis of the
624
percentage of free or adherent bacteria (in blue) and bacteria overlapping with cells (in green)
625
found in blood samples from control and infected mice. The location of bacteria was defined
626
by erosion mask. These data are representative of three independent experiments.
627
28
628
Figure 7. Brucella is found associated with or inside erythrocytes by scanning electron
629
and confocal microscopy. A. Scanning electron microscopy images from blood collected in
630
mice 3h p.i. by i.p. injection of 5x107 CFU of Brucella. Extracellular bacteria (a), attachment
631
of Brucella to erythrocytes (b), deformed erythrocytes suggesting Brucella infection (c-d). B.
632
Confocal microscopy images of blood collected in mice 24h p.i. by i.p. injection of 5x107
633
CFU of Brucella. Attachment of Brucella to erythrocytes (a), Brucella inside erythrocytes
634
(b).
635 636
Figure 8. Brucella is not surrounded by a vacuole in erythrocytes. Transmission electron
637
microscopy analysis of erythrocytes from mice 24h p.i. by i.p. injection of 5x107 CFU of
638
Brucella.
639 640 641 642
29
643
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